Tumor cells of some cancers secrete enzymes that break down the intracellular matrix and invade adjacent tissues. The production of serine protease enzymes such as plasminogen activator enzymes have been observed in connection with the metastasis of tumor cells into healthy tissues. Plasminogen activator, enzymes have been linked to cancer detection, using assays of enzymes extracted from the tumor cells (Camiolo, S. M., Markus, G. and Piver, M. S. "Plasminogen activator content of gynecological tumors and their metastases." Gynecolocical Oncology 26: 364-373, 1987) or assays of supernatants from tissue culture of the tumor cells. (Hasui, Y. et al., "Comparative study of plasminogen activators in cancers and normal mucosa of human urinary bladder." Cancer Research 49: 1067-1070, 1989. Harvey, S. R., et al., "Secretion of plasminogen activators by human colorectal and gastric tumor explants." Clin. Expl. Metastasis 6: 431-450, 1988.)
Urokinase-type plasminogen activator (urokinase or uPA), a serine protease, is not produced in most normal cells, except at low levels in certain types of normal kidney cells, colon and gastric mucosa, and endothelial cells lining small arteries. Urokinase (uPA) can be present in the tissues in several molecular forms,. The inactive proenzyme (scuPA) is composed of 411 amino acids. ScuPa is converted to the active enzyme by cleavage at Lys.158-Ile.159, loss of the Lys.158 and formation of the double chain, high molecular weight form (HMW-uPA) that is 54 kDaltons. A low molecular weight form (LMW-uPA) can also be formed by cleavage of the HMW-uPA at Lys.135-Lys.136 giving a 33 KDa active enzyme.
The active urokinase enzymes convert plasminogen into plasmin, which in turn, dissolves intravascular fibrin blood clots and intracellular fibrin matrix components as well as activating collagenases, laminases, and other related enzymes which are important to the anchorage and growth regulation of cells. Urokinase is produced in a number of tumors such as lung, colon, gastric, uterine, breast, brain cancer and malignant melanoma. High levels of urokinase (&gt;3.5 ug/mg of total protein) extracted from breast tumor tissues is an indicator for high risk of recurrence and shorter patient survival times. Janicke, et al. Fibrinolysis 4:69-78, 1990. Data from these clinical studies showing that a) measurements of uPA in plasma are of no value and that b) measurements of uPA in cytosol fractions give some prognostic value (0.12 benign vs. 1.65 ng/ml in metastatic breast cancer) and c) measurements of the uPA extracted from all of the tumor cells by Triton-X 100 treatment have even more significance (0.23 vs. 3.21.+-.2.40 ng/mg of metastatic breast cancer). However, the standard deviation (S.D.) of these measurements represents .+-.65% for benign breast cancer and .+-.75% for metastatic tissues. Clearly, cell extractions cannot make the precise distinctions that are necessary as to the presence or absence of uPA, since the extractions are from tissues containing some normal cells, some cancer cells that may have no (or very little) uPA and those that may have significant amounts of uPA. (Schmidt, M. et al, "Tumour-associated fibrinolysis: the prognostic relevance of plasminogen activators uPA and tPA in human breast cancer." Blood Coagulation and Fibrinolysis 1: 695-072, 1990). Also cells from primary lung and colon tumor produce more uPA than cells from metastatic tumors, but different methods of extraction and assays give widely variable results (Markus, G. et al., "Plasminogen activator secretion of human tumors in short-term organ culture, including a comparison of primary and metastatic colon tumors." Cancer Research 43: 55-7-5525, 1983.).
It recently has been shown in cancer cells that the HMW active form of urokinase, bound to the tumor cell membrane, is responsible for the local lysis of the extracellular matrix, hence the tissue invasion mechanism for metastasis. (Hollas, W., Blasi, F., and Boyd, D. "Role of urokinase receptor in facilitating extracellular matrix invasion by cultured colon cancer." Cancer Research 51: 3690-3695, 1991.). The unbound uPA and the LMW form is not responsible for local dissolution of extracellular matrix in the immediate vicinity of the metastatic tumor cell. (Cubellis, M. V., Wun, T. and Blasi, F. "Receptor-mediated internalization and degradation of urokinase is caused by its specific inhibitor PAI-1." EMBO Journal 9: 1079-1085, 1990.). Thus the direct measurement of membrane-bound urokinase is more important to the prognostic accuracy than is measurement of all urokinase (membrane-bound and free uPA) present in cells or in the tissue specimen.
Total urokinase has been measured from tumor tissue and secretion by cultured explants. These are difficult to quantitate from biopsy to biopsy, especially if the measurements are made on a large group of cells. The data obtained is an average value of all normal and cancer cells, rather than a measurement of each individual cell. However, direct measurements of intracellular and extracellular urokinase have not been made previously.
A DNA content histogram of normal cells shows a single diploid peak (at G.sub.1 phase) and a tetraploid peak (at G2+M phase). In most tumors, abnormal DNA content of tumor cells is detected as a second G.sub.1 peak or multiple peaks. Abnormal DNA (DNA aneuploidy) is considered as an independent indicator of tumor aggressiveness and poor prognosis that is used to supplement cytopathology grading. Flow cytometry can be used to measure total DNA content as shown in U.S. Pat. No. 4,780,406, Dolbeare et al. entitled Flow Cytometric Measurement of Total DNA and Incorporated Halodeoxyuridine issued Oct. 25, 1988, which is incorporated herein by reference. Cells disassociated and prepared for flow cytometry can be analyzed for cell cycle stage as well as DNA content simultaneously. Flow cytometer measurement of the percentage of proliferating tumor synthesizing DNA (S-phase cells) is. an independent indicator of malignancy. High percentages (15-20%) of S-phase tumor cells usually indicates an aggressive malignancy and usually correlates well with abnormally high DNA content. The labeling index (LI) obtained by pulse-labeling cells with DNA precursors represents the rate that DNA is being synthesized in tumor cells. Usually, a LI&gt;4% is associated with a higher probability of recurrent malignancy. (Merkel, D. E. Dressler, L. G. and McGuire, W. L., "Flow cytometry, cellular DNA content and prognosis in human malignancy." J. Clin. Oncology 5: 1690-1703, 1987.)